Reduction-alkylation strategies for the modification of specific monoclonal antibody disulfides

Abstract

Site-specific conjugation of small molecules and enzymes to monoclonal antibodies has broad utility in the formation of conjugates for therapeutic, diagnostic, or structural applications. Precise control over the location of conjugation would yield highly homogeneous materials that could have improved biological properties. We describe for the first time chemical reduction and oxidation methods that lead to preferential cleavage of particular monoclonal antibody interchain disulfides using the anti-CD30 IgG1 monoclonal antibody cAC10. Alkylation of the resulting cAC10 cysteine thiols with the potent antimitotic agent monomethyl auristatin E (MMAE) enabled the assignment of drug conjugation location by purification with hydrophobic interaction chromatography followed by analysis using reversed-phase HPLC and capillary electrophoresis. These analytical methods demonstrated that treating cAC10 with reducing agents such as DTT caused preferential reduction of heavy-light chain disulfides, while reoxidation of fully reduced cAC10 interchain disulfides caused preferential reformation of heavy-light chain disulfides. Following MMAE conjugation, the resulting conjugates had isomeric homogeneity as high as 60-90%, allowing for control of the distribution of molecular species. The resulting conjugates are highly active both in vitro and in vivo and are well tolerated at efficacious doses.

1

Conjugation strategy. The drug-linker vcMMAE reacts with a mAb cysteine to form the ADC. The potent antimitotic agent MMAE is released from the ADC following proteolysis. As many as 8 molecules of vcMMAE can react with each mAb following reduction of the 4 interchain disulfides present in cAC10.

2

Composition of E4 mixture as a function of conjugation methodology. HICHPLC chromatograms were integrated to determine the abundance of E0, E2, E4, E6, and E8 in the mixtures. Values are ± standard deviation for 4 (DTT partial reduction), 3 (TCEP partial reduction), or 6 (DTNB partial reoxidation) separate experiments. The contributions from odd-loaded species, typically less than 10%, are not shown. The sum of E0, E2, E4, E6, and E8 was normalized to 100%. The simulated reduction assumes that the rate constants for reduction of the 4 interchain disulfides are equal for a random reduction process, and the sum of E0, E2, E4, E6, and E8 equals 100%.

3

Analytical characterization of ADCs. (A-D) Reversed-phase HPLC and (E-H) capillary electrophoresis were used to analyze (A, E) E4 mixture made by partial DTT reduction followed by MMAE conjugation; (B, F) E4 mixture made by DTNB partial reoxidation followed by MMAE conjugation; (C, G) E4 made by partial DTT reduction followed by MMAE conjugation and purified by preparative HIC ; and (D, H) E4 made by partial DTNB reoxidation followed by MMAE conjugation and purified by preparative HIC. HPLC injections were 20 μL of 1 mg/mL cAC10-vcMMAE treated with 20 mM DTT for 15 min at 37 °C. Separations were performed at 80 °C. Capillary electrophoresis was performed under non-reducing conditions.

4

Potential isomers for E2, E4, and E6. Isomers with one heavy-heavy disulfide (2A, 4A, and 6A) can be further subdivided into isomers with either the first or the second disulfide intact, but because the analytical methods described herein cannot distinguish these subgroups, only one isomer is shown. Below the isomer are the chain compositions under denaturing conditions (first line, non-reducing; second line, reducing). For denaturing and non-reducing conditions, the possible species formed are L, H, HL, HH, HHL, and LHHL, where the remaining interchain disulfides link the indicated chains. For denaturing and reducing, the possible species formed are L0, L1, H0, H1, H2, and H3, in which the numbers indicate how many drug molecules are attached to the light or heavy chain. The locations of MMAE conjugation are indicated by stars. Unconjugated cAC10 (E0) and fully conjugated cAC10 (E8) are not shown.

5

Composition of isomeric population of purified E2, E4, and E6. The percent of the E2 and E6 isomers were determined from reversed-phase HPLC data using Equations 1 and 2, respectively. The percent of E4A was determined from capillary electrophoresis data using Equation 3 and then the percent of E4B and E4C were determined from reversed-phase HPLC data using Equations 4 and 5. For each drug loading level, the sum of the isomers is equal to 100%.

6

Simulation of random reduction reaction. cAC10 reduction was simulated with equal rate constants for reduction of heavy-heavy and heavy-light disulfides. (A) The simulated drug loading abundance is shown with bars for the reduction of an average of 2 disulfides to yield an average of 4 antibody cysteines. The percent differences between the experimentally determined drug loading abundance from HIC-HPLC data for DTT and TCEP partial reduction shown in Figure 2 and the simulated values are shown in lines. (B) The simulated isomer distribution is shown in bars. The percent differences between the experimentally determined isomer abundances shown in Figure 5 and the simulated values are shown in lines. In both panels, a positive number indicates that the experimental data is greater than the simulated value, while a negative number indicates that the experimental data is lower than the simulated value.

7

Single dose efficacy of E2 and E4. SCID mice with Karpas 299 tumors were treated with single doses of (A) E2 made by DTT partial reduction and DTNB partial reoxidation and (B) E4 made by DTT partial reduction, TCEP partial reduction, and DTNB partial reoxidation. Animals were considered cured if they were tumor free 101 days post tumor implant.